The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of materials of interest that may be readily and accurately determined, as well as the methods for the determination. Over the last several decades, testing for numerous substances such as drugs of abuse, or other biological molecules of interest has become commonplace. In recent years, immunoassay based on the interaction of an antibody with an antigen has been extensively investigated for this purpose.
Based on the unique specificity and high affinity of antibodies, an immunoassay can accurately and precisely quantitate substances at the very low concentrations found in biological fluids. Immunoassay can be categorized according to its design. If the bound (to antibody) and the unbound antigens (or analytes) are not physically separated during the assay, it is called a homogeneous assay; otherwise, a heterogeneous assay. Immunoassays can use either labeled antigen or label antibody. The assay can be a competition assay (frequently used in small molecule detection; typically limited reagents are used), or a sandwich assay (for large molecules such as proteins; usually excess reagents are used). Depending on the signal generation and detection system utilized, immunoassay can be a turbidity or a nephelometry assay, a radioimmunoassay, a calorimetric assay, a fluorescence assay, a chemiluminescence assay, an enzyme-labeled assay (the signal may be measured by colorimetry, fluorometry, chemiluminescence, or bioluminescence, etc.), an electrogenerated chemiluminescent assay (ElectroChemical Luminescence or ECL assay), a target amplification assay (using methods such as PCR to amplify the target), or a signal amplification assay (via enzyme channeling or use liposome carrying a bag of signal, for example), etc.
By way of further background, a hapten may be defined as a chemical composition of limited molecular weight (usually less than 1000) which in and of itself does not elicit antibody formation when introduced into a host animal. However, when covalently bonded to a high molecular weight antigenic carrier, the resultant hapten-carrier conjugate can elicit in the host animal the formation of antibodies which recognize the hapten composition. Examples of haptens to which antibodies have been raised in this fashion are numerous, including such classes of materials as medicaments (therapeutic drugs), drugs of abuse, amino acids, dietary supplements, and metabolites, small peptides, steroids, hormones (such as thyroxine), and aromatic residues such as the dinitrophenyl moiety. Typical carriers are large polyvalent molecules such as proteins, polysaccharides and glycoproteins which are not native to the host animal. The methods for preparation of hapten-carrier conjugates are well known in the art and have been reviewed (B. F. Erlanger, Methods in Enzymology, v. 70, p. 84, 1980).
S-adenosyl methionine, commonly known as SAM, or SAM-e, or AdoMet, is a natural compound found in all living cells. It is one of the most used enzymatic substrates in biochemical reactions, second only to the universal energy storage and transfer molecule, adenosyl triphosphate (ATP).
The chemical structure of SAM is:

SAM plays a crucial role in the process called transmethylation. Methylation is involved in nearly every aspect of life. SAM is the primary “methyl” donor for a variety of methyl-transfer reactions in DNA, RNA, proteins, lipids, and small molecules in the body.
Proper DNA methylation is essential for normal embryonic development. Methyl-transferase gene homozygously deleted (knocked out) has been proven lethal (Pegg, A. E., Feith, D. J., Fong, L. Y., Coleman, C. S., O'Brian, T. G., and Shantz, L. M., 2003, Biochem. Soc. Trans. 31, 356-360). DNA improperly methylated has been found in many tumors. Alterations in DNA methylation patterns induce the expression of oncogens or silence the expression of tumor suppressor genes, and methyl deficient diets have been shown to promote liver cancer in rodents. Methylation of DNA results in protection of the genome from restriction enzymes (Loenen, W. A. M., 2003, Nucleic Acids Res., 31, 7059-7069; Murray, N. E., 2000, Microbiol Mol Biol Rev, 64, 412-434). SAM has been shown to control gene expression by binding to structural domains embedded within the non-coding region of certain mRNAs (Corbino, K. A., Barrick, J. E., Lim, J., Tucker, B. J., Pusharz, I., Mandal, M., Rudnick, N. D., and Breaker, R. R., 2005, Genome Biol., 6, R70).
SAM provides the methyl group in the production of essential bio-molecules such as carnitine (the fat burner), acetyl-L-carnitine (the neuronutrient and membrane transporting agent), phosphocreatine (the primary ATP reservoir), epinephrine/adrenalin (the endogenous catecholamine, stress hormone and neurotransmitter), phosphatidylcholine (the most important membrane phospholipids), and melatonin (circadian rhythm modulator), etc.
In addition to transmethylation, SAM also plays a myriad of roles in other metabolic pathways. The transsulfuration begins with S-adenosylhomocysteine (SAH), the residual structure of SAM upon donating the methyl group (transmethylation). Hydrolysis of SAH yields homocysteine, which in turns converts to cystathionine, then cysteine, and eventually, to glutathione, the hepatocellular antioxidant and life-saving detoxification agent. Since dietary cysteine content is low, and up to 80% of dietary cysteine may lose its sulfhydryl groups through gastrointestinal tract, SAM is the main providing source of cysteine, the building block of glutathione.
The aminopropylation is another process initiated with SAM through decarboxylation. The decarboxylated SAM then couples with putrescine to generate spermidine and spermine which are critical to cell growth, differentiation and the stability of DNA and RNA. Furthermore, Methylthioadenosine (MTA), the by-product of polyamine synthesis, is a powerful analgesic and anti-inflammatory agent. This may be, at least partially, responsible for the clinical benefits observed in the treatment of osteoarthritis, rheumatoid arthritis and fibromyalgia with SAM (G. Stamentinoli, 1987, Pharmacologic aspects of SMe, Am J Med, 83 (suppl 5A), 35-42; C. di Padova, 1987, SAMe in the treatment of osteoarthritis, Am J Med 83 (supp 5A), 60-65; A. Tavoni et al, 1987, evaluation of SAMe in Primary Fibromyalgia, Am J Med, 83 (suppl 5A), 107-110).
More recently, it has been recognized that SAM, in the presence of Vitamin B12, gives rise to 5′-deoxyadenosyl radical which generates other radicals on the enzyme methionine synthetase or coupled enzymes (Toohey, J. I., Biofactors, 2006, 26(1) 45-57; Kozbial, P. S. and Mushegian, A. R., 2005, BMC Struct Biol, 5, 19.) The free radical propagation results in rearrangement or removal of toxic substances accumulated. One example is conversion of homocysteine to iso- or β-methionine, which is readily degraded to methanethiol.
Poor methylation or deficiency of SAM has been implicated or related to the development of birth defects, cardiovascular disease, cancer, liver disease, neurological dysfunction, mood swing, herpes outbreak, diabetes, depression, indigestion, chronic fatigue, and age-related illness such as Alzheimer's disease, etc.
Treatment with SAM has been proven to be as effective as prescription tricyclic antidepressants, non-steroidal anti-inflammatory drugs (NSAIDS), and showed efficacy in treatment of some liver conditions such as cholestasis of pregnancy and intrahepatic cholestasis associated with liver diseases (Healthcare Research and Quality, Dept of Health and Human Services). More significantly, SAM is well tolerated and no serious side effects have been noted. This is in contrast to those frequently associated with prescription antidepressants (e.g., headaches, weight gain, and sexual dysfunction, etc.) or NSAIDS (e.g., irritation and damage of gastrointestinal linings, even internal bleeding upon chronic use, and increase risk of heart disease). It also provides rapid onset of relief in comparison to the tricyclic antidepressant treatment. Additional benefits and claims have been published in both scientific literature and popular reporting, although further studies and confirmation were awaited. Usage of SAM by patient who suffers from bipolar disease is contraindicated for risks of exacerbation into manic depression.
It has been reported that SAM in the tissues of older rats is significantly lower that of the younger animals. Similar findings were reported in human with aging, dementia; liver disease, alcoholism, and depression (R. Baldessarini, 1997, Neuropharmacology of SAMe, Am J Med 83 (suppl 5A), 95-103) The importance of S-adenosylmethionine as a biomarker is without question. It may well be a vital link between health and disease, from birth to mature to prime and then to age.
In Europe SAM has been classified and sold as a drug (as Ademetionine) for treatment of depression, liver disorders, osteoarthritis and fibromyalgia since 1979 in Italy, since 1985 in Spain, and since 1989 in Germany (Teodoro Bottiglieri, Am J Clin Nutr 2002, 76 (suppl) 1151s-1157s.) In the United States, SAM is marketed as a dietary supplement for the comfort of bone and joint, support of liver health, and well being of mood since 1999. It has quickly become one of the most popular dietary supplements sold among the estimated 30,000 dietary items in the market. The regulation on dietary supplement is substantially less in comparison to drugs or pharmaceuticals. In view of being the primary methylating agent capable of modifying DNA, RNA, protein, and many functional molecules in the body it is critically important that scientific information is accurately documented and accessible. Guidelines and oversight on its use are in place. However, despite of the so far publicized safety record, clinical trials of SAM are mostly short-term studies, and generally involved only small number of patients. Information regarding to drug or food interactions with SAM is extremely limited.
Furthermore, neither the normal range of SAM concentration in blood/serum/plasma nor the optimal concentration had been extensively discerned and determined. Due to various reasons including patient population, sample handling, pre-treatment, and detection methods, etc., published results were vastly inconsistent. Even from the same laboratory, utilizing similar technique, wide ranges of concentration have been reported for healthy patients. The data are in general most useful for comparison within the context of the particular studies.
Since SAM is an intrinsically unstable molecule, and its optical density maximum of 258-260 nm is not a distinguished absorption, the determination of its concentration in various biological fluids and tissues has always been a challenging task. A simple, convenient method that does not require costly instrumentation (LC, MS, and combinations) is clearly desirable for the determination of the biological concentration of SAM, and to monitor its change and metabolic paths in the body fluids, tissues and organelles. It is also critically necessary to understand the impact and consequence when SAM is been used daily as a nutritional supplement today.
The SAM concentration in the blood, serum or plasma of a healthy adult (or any age group) has not been established despite the fact that there have been substantial efforts and interests in determining the concentrations in various tissues or biological fluids, and in different health and disease conditions. Largely due to the absence of a reference standard for SAM itself, and the lack of an effective method to measure SAM from a biological sample, almost all studies utilize internal standard and the reported concentrations between studies varies greatly for healthy individuals as well as those in disease state.
The majority of SAM in blood is found in erythrocytes, with concentration at μM level (typically between 2 to 4 μM). Its concentration in serum or plasma for healthy adults is reported in the neighborhood of 60-150 nM. Variations in concentrations in these ranges continued to be reported from time to time perhaps due to problems such as instability of SAM, the lack of standardization of SAM and the sample handling issues. The concentration in CSF appears to be higher than that in serum, at between 100 to 250 nM. The urine SAM concentration has been reported in the range of 20-150 μM.
In view of the importance of SAM, it is desirable to have an easy and reliable method to measure its concentration in a biological sample. Assay range and sensitivity required for measuring SAM in most common biological samples for both normal and abnormal concentrations are estimated as follows:
Serum/Plasma 20-300 nM(2.0 × 10−8 to 3.0 × 10−7 M)assay-Erythrocytes600-8000 nM(6.0 × 10−7 to 8.0 × 10−6 M)assay-Whole blood300-4000 nM(3.0 × 10−7 to 4.0 × 10−6 M)assay-CSF assay- 30-500 nM(3.0 × 10−8 to 5.0 × 10−7 M)Urine assay-  5-300 μM(5.0 × 10−6 to 3.0 × 10−4 M)Liver tissue 20-200 nmole/g tissueassay-Cells assay-  1-200 ng/106 cells
A classical assay method for measurement of SAM in rat liver utilized the tripolyphosphatase activity that was associated with S-adenosylmethionine synthetase (Y. Suma, et al, J. Biochemistry, 96, 679-682, 1984.) in rat liver. The tripolyphosphatase activity is stimulated by low concentrations of S-adenosylmethionine (Mudd, S. H., 1963, J. Biol. Chem. 238, 2156-2163.) The assay sensitivity was reported at 0.1 nmole of SAM in an assay volume of 0.1 ml (i.e., 10−6M). Samples were lyophilized, homogenized in acid, and centrifuged. The supernatant was then passed through Dowex 1 (HCO3− form) to rid of endogenous inorganic phosphate and other potential interferents in the tissue. Great care has to be taken to avoid inorganic phosphate contamination from all reagents including the enzyme preparation, as well as glasswares.
The more common method for measuring SAM in tissues or biological fluids is HPLC or electrophoresis after sample preparation normally encompassing the protein precipitation and/or extraction (P. Giulidori and G. Stramentinoli. 1984, Anal. Biochem. 137: 217-220; Loehrer, F. M., et al. Nephrol Dial Transplant, 1998, 13: 656-661; Melnyx, S. et al. Clin Chem, 2000, 46: 265-272; E. S, Struys, et al. Clinical Chemistry, 2000, 46 (10): 1650-1656; A. Becker, et al., European J. Clin. Invest., 2003, 33: 17-25). Post column detection may include derivatization, then measurement through absorption, fluorescence, or electrochemical change, and more recently by Mass Spectrometry (MS), or Tandem Mass Spectrometry (MS/MS). Radioisotopes or stable isotopic molecules of SAM are frequently used for internal reference purpose. These methods are capable of measuring low level of SAM in serum or plasma; however, the process typically is laborious, time consuming and/or requires expensive equipments. Another drawback is that it usually does not distinguish the diastereoisomers of SAM at the sulfonium position.
SAM is produced biologically in the (S,S) configuration at the sulfonium and α-amino acid carbon, respectively. Under normal physiological conditions or storage conditions, SAM spontaneously racemizes to form a mixture of (R,S) and (S,S) isomers. Most methyltransferases are reported to be specific to the (S,S) form of SAM only.
A stereospecific colorimetric assay of SAM based on an enzyme-coupled reaction, thiopurine methyltransferase-catalyzed thiol methylation, has been developed by Sunny Zhou's group at Washington State University at Pullman, Wash. (Cannon, L. M, et al, Analytical Biochemistry, 308 (2) 358-363, 2002). The assay utilizes stereo-specific characteristics of a recombinant human thiopurine S-transmethyltransferase (TPMT, EC 2.1.1.67) and measures the change of absorbance at 410 nm of 2-nitro-5-thiobenzoic acid (TNB) vs. 2-nitro-5-methylthiobenzoic acid. The downside of the assay is that both the starting material and the product exhibits 410 nm absorption, and higher absorption associated with the starting material; therefore, the assay is effectively measuring the decrease in absorption at 410 nm. This is potentially complicated by the fact that TNB can be easily oxidized to form the disulfide, 5.5′-dithiobis-2-nitrobenzoic acid, which also has a low 410 nm absorption.
An NMR methodology was described for the determination and characterization of dietary supplement SAM without pure reference standards. A 400 MHz spectrometer is used to assess chemical structure, differentiate and determine the ratio of (S,S) and (R,S) diastereomers (G. M. Hanna, 2004, Pharmazie, 59 (4), 251-256.) The study of 10 synthetic lots showed (S,S) content ranges from 0 to 82.3%.
Recently, the necessity of differentiate diastereoisomers of SAM has been drawn to question. One study showed that both isomers gave significant activities, in terms of functional outcome, in increasing blood flow and bile production in isolated perfused rat livers (Tredger, J. M. SAMe in ischemia-reperfusion injury: experimental basis and clinical findings. 4th reunion of metabolism of methionine, Sierra Nevada, Granda, Spain, 1998; Dunne, J. B., Alexander, B., Williams, R., Tredger, J. M, and Hoffman, J. L., Evidence that SAMe diastereoisomers may reduce ischaemia-reperfusion injury by interacting with purinoceptors in isolated rate liver. Chromatographyic analysis of the chiral and covalent instability of S-adenosylmethionine. Br J Pharmacol, 1998, 125: 225-233). It is not clear whether enzymes other than methyltransferase that involved SAM are responsible for the functional activity, or a reversible racemization is contributing to this result.
Perhaps the notion that all biological methylations uses only the (S,S) diastereomer of SAM requires a more thorough examination as well. From literature we found that homocysteine S-methyltransferase has been reported as an exception capable of utilizing both diastereomers with respects to its sulfonium ion center. (J. Durell et al, 1957, Biochim. Biophys. Acta, 26, 270; V. Zappia et al, 1969, Biochim. Biophys. Acta, 178, 185.) Another recent study revealed a homocysteine methyltransferase from yeast recognized both (R,S) and (S,S) isomers; in fact one yeast preferred the (R,S) isomer as substrate, while another utilized the (R,S) isomer exclusively. The (R,S)-specific homocysteine methyltransferase activity was also shown to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans (C. R. Vinci and S. G. Clarke, Recongition of Age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae, 2007, J. Biol. Chem., 282 (12), 8604-8612).
A commercial assay (for R&D purpose only), Bridge-It® S-adenosylmethionine fluorescence assay has emerged in 2007 (Mediomics, LLC, St Louis, Mo.). In the presence of SAM the activity of a unique DNA sequence-specific MetJ protein binding to DNA increases. The MetJ consensus sequence was split into two DNA “half-sites”. One fragment is labeled with fluorescein and the other with Oyster® 645 fluorophore. The concentration of SAM is in direct proportion to the formation of DNA-MetJ protein complex, which resulted in two fluorophores in close proximity and allowed energy transfer to occur (Excitation at 485 nm for fluorescein and emission at 665 nm for Oyster® 645.). A dynamic range from 0.5 μM to 20 μM and detection limits of 0.5 μM were claimed.
U.S. Pat. No. 6,713,273 discloses a method for measuring SAM utilizing a recombinant S-adenosylhomocysteinase (S-adenosylhomocysteine hydrolyase, SAHH, EC 3.3.1.1) coupling with glycine N-methyltransferase (EC 2.1.1.20) and homocysteine α,γ-lyase (rHCYase). The methyltransferase converts SAM to SAH which is then hydrolyzed by SAHH to generate homocysteine. If rHCYase is also included, the homocysteine can be converted to hydrogen sulfide, α-ketoglutarate and ammonia. Either the amount of hydrogen sulfide or homocystiene generated is in direct proportion to SAM concentration in the sample. The idea of enzyme channeling is sound, but no actual example was demonstrated to show that this process indeed worked with a biological sample. An approach like this has a potentially risk of interference by the presence of endogenous compounds such as homocysteine which concentration in serum is within 5-20 μM range, an amount substantially higher than that of SAM in serum or plasma (50-150 nM).
US published patent application 2005/0003378 based on provisional application Ser. No. 60/434,397 discloses an in vitro assay for SAM as a demethylase inhibitor.
Another molecule of interest, S-Adenosylhomocysteine (SAH), is the precursor leading to the biosynthesis of SAM, as well as the product of all transmethylation reactions involving SAM as the methyl donor; i.e., SAH is metabolically linked to SAM, and structurally it contains a single carbon (as methyl) less than SAM. The co-existence and structure similarity of SAM and SAH present a great challenge to develop a method for the specific determination of the concentration of either molecule in a biological sample. The unstable (highly reactive) nature of SAM renders the level of difficulty for its determination even higher.
As the immediate precursor of all of the homocysteine (HCys) produced in the body, SAH has been raised as a possibly more sensitive indicator for the risk of vascular disease than plasma HCys recently (C. Wagner and M. Koury. Am. J. Clin Nutr 2007, 86: 1581-1585; D. Kerins, et al. Am. J. Clin Nutr 2001, 74: 723-729). The total plasma concentration of SAH is normally in the range of 15-30 nM (by contrast, plasma homocysteine is in the range of 5-15 μM). Like SAM, with no distinguished absorption, the determination of SAH in serum or plasma has been a challenge. Advanced method such as LC with post column derivatization, HPLC-MS, or HPLC-MS/MS with internal reference is a recent development for its determination. However, these methods typically involve expensive instrumentations, laborious sample preparation, and time consuming procedures. Unlike SAM, however, SAH is a relatively stable compound; the sample handling and stability are usually non-problematic.
An enzymatic method to quantify SAH in biological samples has been suggested in the art. Mutated or genetically engineered S-adenosylhomocysteine hydrolase which retains or has enhanced binding affinity toward either Hcys or SAH, and with attenuated catalytic activity has been identified or developed (U.S. Pat. No. 6,376,210; Y. Tran, et al. Clinical Chemistry, 46:1686-1688, 2000)
An antibody specific to SAH has been previously developed for the determination of serum/plasma homocysteine. In such a homocysteine assay, homocysteine was first converted to SAH via the reversible enzyme S-adenosylhomocysteine hydrolase (EC 3.3.1.1] in the presence of adenosine. Anti-SAH antibody was then used to competitively capture SAH in the assay (Homocysteine assay by Abbotts Laboratory; Homocysteine Assay by Axis-Shield ASA, Norway). Since the serum/plasma concentration of homocysteine is at 5-20 μM range, the assay sensitivity requirement for the enzymatically converted SAH is in the same ballpark. The presence of endogenous SAH (at tens nM concentration) is not an issue, neither is the cross-reactivity of the anti-SAH toward SAM because its concentration is also a couple orders of magnitude lower.
Recently an immunoassay for SAH in plasma has been reported by A. Capdevila, et al. (J Nutritional Biochemistry, 18: 827-831, 2007) utilizing Axis-Shield's anti-SAH antibody and SAH conjugate. Although the initial study of the antibody indicated some cross-reactivity toward SAM, the paper claimed SAM was not recognized even at high concentration under the current assay conditions.
Also, since SAH is the product of all methylation reactions involving SAM as methyl donor, increased concentration of SAH (or [SAH]) in tissues are frequently accompanied by decreased concentration of SAM ([SAM]). Therefore, the ratio of [SAM] and [SAH] may be a more sensitive indicator than the concentration of either SAM or SAH alone, particularly when their changes are subtle at early stages of dysfunction or abnormal conditions. The ratio of the concentration of SAM to the concentration of SAH known as “the methylation index,” was first proposed by Cantani, et al. as an indicator of the methylating capacity of the cell (Inhibition of S-adenosylhomocysteine hydrolase and their role in the regulation of biological methylation. In Usdin E., Borchardt R T, Creveling C R, eds. Transmethylation. New York, N.Y. Elsevier North Holland, 1978, pages 155-164.) The ratio was later referred by M. S. Hershfield et al as “methylation index” in Cancer Res 43: 3451-3458, 1983.
Furthermore, normal SAM concentration in plasma appears to be different, greatly depending on gender (normally, men>women), individual's weight, and maybe ethnicity, and diet, etc. Similarly dependency exists for SAH concentrations since SAM and SAH are closely tied together metabolically. By utilizing the ratio of [SAM] and [SAH] it is likely these variables can be eliminated or diminished.
Connection of SAM and SAH to cardiovascular disease, depression, cancer and aging-related diseases such as Alzheimer's disease is well documented. Methylation is highly critical in fetus development, in differentiation, in epigenetic regulation of protein expression via DNA and the histone methylation. The valuation of the S-adenosylmethionine and methylation capacity index is in their scientific basis as “vitality” indicators or “wellness” markers, as opposed to homocysteine (or S-adenosylhomocysteine as some would prefer to use) as a “disease” marker.
Combining a method (e.g., an immunoassay) for quantifying SAH with the immunoassay described in the present invention will allow the determination of the so-called “methylation index”, a very rapid and simple task that every medical research facility and clinical laboratory can perform.